For the analysis of large molecules, e.g. biomolecules, mass spectrometry with ionization by matrix-assisted laser desorption and ionization (MALDI) has become a standard method. For the most part, time-of-flight mass spectrometers (TOF-MS) are used for this purpose, but ion cyclotron resonance spectrometers (FT-ICR=Fourier transform ion cyclotron resonance) as well as high-frequency quadrupole ion trap mass spectrometers can be applied here. Normally, the biomolecules are in an aqueous solution. In the following, the high molecular weight substances including the biosubstances, the molecules of which are to be analyzed, are called "analytes".
The term biomolecules or biosubstances here mainly denotes the oligonucleotides (i.e. the genetic material in its various forms such as DNA or RNA) and the proteins (i.e. the essential building blocks of the living world), including their particular analogs and conjugates, such as glycoproteins or lipoproteins.
The choice of a matrix substance for MALDI is dependent upon the type of biomolecules; many more than a hundred different matrix substances have become known in the meantime. The task of the matrix substance is to separate the sample molecules from each other, to bond them to the sample support, to transform them into the gas phase during laser bombardment by the formation of a vapor cloud without destroying the biomolecules and if possible without attachment of the matrix molecules, and finally to ionize them there under protonation or deprotonation. It has proven favorable for this task to incorporate the analyte molecules in some form into the usually crystalline matrix substances during their crystallization or at least into the boundary-surfaces between the small crystals.
Various methods are known for applying the sample and matrix. The simplest of these is the pipetting of a solution with sample and matrix onto a clean, metal sample support plate. The solution drop is wetting on the metal surface an area, the size of which corresponds approximately to the diameter of the drop and is dependent on the hydrophilia of the metal surface and the characteristics of the droplet. After the solution dries, the sample spot consists of small matrix crystals spread over the formerly wet area, whereby generally there is no uniform coating of the wetted area. In aqueous solutions, most of the small crystals of the matrix generally begin to grow at the margin of the wet area on the metal plate. They grow toward the inside of the wet area. Frequently they form long crystals in radial direction, such as 5-dihydroxybenzoic acid (3-DHB) or 3-hydroxypicolinic acid (3-HPA), which peel off of the support plate toward the inside of the spot. The center of the spot is frequently empty or covered with fine small crystals which are however hardly utilizable for MALDI ionization due to the high concentration of alkali salts. The analyte molecules are irregularly distributed. This type of coating therefore demands visual observation of the sample support plate surface through a video microscope, which can be found on all commercially manufactured mass spectrometers for this type of analysis. Ion yield and mass resolution fluctuate in the sample spot from site to site. It is often a troublesome process to find a favorable location on the sample spot with good analyte ion yield and good mass resolution, and only experience and experimentation have been helpful here up to now.
For matrix substances which dissolve only very poorly or not at all in water, such as .alpha.-cyano-4-hydroxycinnamic acid, it has proven favorable to create a very thin layer of crystals on the surface before applying the aqueous analyte solutions, for example by applying a solution of matrix substance in acetone. This type of MALDI coating is very successful for peptides (O. Vorm et al., J. Am. Soc. Mass Spectrum., 5, [1994], 955). In particular, the coating demonstrates site-independent sensitivity in the sample spot, a basic prerequisite for any automation of the analysis. Unfortunately, this type of homogenous preparation cannot be used for water soluble matrices, such as for oligonucleotides, for which 3-hydroxipicolinic acid (3-HPA) in an aqueous solution has proven to be the most favorable matrix up to now. However, this matrix demonstrates the edge effects described above in an extreme manner.
A favorable method for oligonucleotide sample loading is performed on silicon chips. The oligonucleotides bonded to the surface of the chips are bombarded with microdroplets of matrix solution (3-HPA) of only a few hundred picoliters using a piezo-operated micropipette, whereby a crystal structure with uniform MALDI sensitivity is generated (D. Little et al., paper presented at the 45th ASMS Conference on Mass Spectrometry and Allied Topics, Palm Springs, Jun. 2-5, 1997).
As described in patent DE 196 28 178, many sample spots can be applied in pipette robots onto a sample support with a high density using multiple pipettes through repeated transfer of samples from microtiter plates. However, the location accuracy of the sample spots is dependent on the precision of the sample robot. Commercially available sample robots, however, only have a mechanical precision of 200 micrometers at best. This application, without special preparation technique, leads to the above described irregular sample spots.
Even the methods for the MALDI technique indicated in the patent applications DE 196 17 011 and DE 196 18 032, using nitrocellulose, have not proven successful up to now for water soluble matrices and in particular for oligonucleotides.
But even when applying very small sample spots of reproducible sensitivity, it is a troublesome method to precisely determine the coordinates for the sample spots in the mass spectrometer, using only mass spectrometric means without any other auxiliary devices, if they have been applied inaccurately. Especially for a high sample throughput, it is therefore extremely desirable to know the location of the sample spots as exactly as possible before analysis. Only then fast automation becomes possible, meaning analysis of many samples without continuously performing control measurements. Especially advantageous would be application of the sample spots in a precise grid.
For a high sample throughput, automation of all analysis steps, including the preparation of the samples, is necessary. While sample preparation in pipette machines can proceed today very well automatically, the heterogeneity of MALDI preparations with water soluble matrices and the imprecise application of sample spots still strongly preclude automation of mass spectrometric measurement.
It is the objective of the invention to find a type of sample support enabling sample preparations which allow for automation of the mass spectrometric MALDI analyses of large molecules, especially biomolecules, forming precisely located sample spots with reproducible ionization yield. The sample spots should be arranged in a precisely located array, even if droplets are applied with a pipette robot that operates less precisely. Methods for favorable manufacture of such sample support plates and for loading the plates with samples must be found.